CFTR on an Automated Patch Clamp System

Abstract

One of the significant challenges for Automated Patch Clamp (APC) instruments has been to measure leak ion channels such as Cystic Fibrosis Transmembrane conductance Regulator (CFTR). CFTR functions as a chloride channel and controls the regulation of different transport pathways. Mutations in the CFTR gene have been found to cause cystic fibrosis and congenital bilateral aplasia of the vas deferens. Forskolin is normally used to activate the CFTR channel. Forskolin activates adenylate cyclase, which cause ATP to make cAMP, which then works on the cAMP-dependent protein kinase (PKA). PKA phosphylates the CFTR channel and thereby opens the channel. Fluoride can stimulate CFTR Cl- channel activity, by stimulating the adenylate cyclase. The activation of the CFTR channel by Fluoride is important, since many APC platforms based on planar chip technology use Fluoride in the intracellular saline solution, as a way to obtain sufficiently tight seals. CFTR channels show little or no voltage dependence, and can be challenging to leak compensate. Several different leak methods can be utilized on a QPatch, but sweep subtraction would be preferred. With sweep subtraction a high dose of relative specific antagonist is added, and deduct the antagonist blocked current from the rest of the experiments. Data from human CFTR channels demonstrate that the Sophion QPatch is capable of performing accumulated dose response experiments on leak channels, and that sweep subtractions is a useful tool in APC systems.